Rho family small GTPases are molecular switches involved in the regulation of diverse cellular functions including various cytoskeleton related events and gene transcription. They may act as critical links to many aspects of cancer development. The Rho GTPase-activating proteins (RhoGAPs) are one of the major classes of regulators of Rho GTPases found in all eukaryotes that are essential in cell cytoskeletal organization, growth, differentiation, and neuronal development, and in certain cases may behave as tumor suppressors. Recent studies have implicated them as specific negative regulators of Rho protein pathways and provided clues on how the RhoGAP-catalyzed GTPase-activating reaction might proceed. The primary purposes of this study are to delineate the mechanism of substrate specificity and efficacy, to understand the mode of regulation, and to investigate the signaling roles of RhoGAP members. In addition, the possible use of RhoGAPs as downregulators of specific Rho GTPases in reversing cancer cell phenotypes will be explored. In specific aim 1 we propose to dissect the mechanism of specificity and efficiency of RhoGAP domain based on the available structure-function information of RhoGAPs. In specific aim 2 we will attempt to determine how various intracellular signals, through kinases and/or SH3 domain, might converge on RhoGAPs leading to their tight regulation, in specific aim 3 we will examine the normal function of two RhoGAPs, Cdc42GAP and p200RhoGAP, by expression, RNAi knockdown and gene targeting knockout approaches. Finally, we will develop a set of novel, RhoGAP-based molecular tools that can be utilized for specific downregulation of individual Rho GTPase activities and test their anti-tumorigenic potential in a variety of cancer cells in which Rho GTPase signaling is altered. These studies will provide mechanistic insights into the regulatory function of RhoGAPs in cell signaling and may generate therapeutic reagents targeting Rho GTPase pathways in human cancers.